University of Texas Southwestern Medical Center at Dallas and University of Houston, Houston, Texas.

Abstract

OBJECTIVE:

Current treatment options for lupus are far from optimal. Previously, we reported that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin, MEK-1/ERK-1,2, p38, STAT-3, STAT-5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were overexpressed in splenic B cells in an age-dependent and gene dose-dependent manner in mouse strains with spontaneous lupus. Since the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK-1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis.

METHODS:

The synthetic triterpenoid CDDO-Me or placebo was administered to 2-month-old B6.Sle1.Sle3 mice or MRL/lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease.

CONCLUSION:

These findings underscore the importance of AKT/MEK-1/2/NF-κB signaling in engendering murine lupus. Our findings indicate that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematologic, autoimmune, and pathologic manifestations of lupus.

CDDO-Me attenuates disease in B6.Sle1.Sle3 spontaneous lupus mice. 2-month-old female B6.Sle1.Sle3 (N = 20/group) were treated with CDDO-Me or placebo (sesame oil) as indicated. CDDO-Me ameliorated splenomegaly (A-B) and suppressed expansion of activated CD4+ T cells in B6.Sle1.Sle3 mice examined 60 days following treatment (C). The flow cytometry plots shown in the figure are from one representative experiment, while data from all experiments are summarized in . H&E staining of kidney shown is representative of several similar experiments (D, n = 10). Proteinuria (E), glomerulonephritis scores (F) and BUN (G) were reduced in CDDO-Me-treated B6.Sle1.Sle3 mice after two months of treatment. CDDO-Me treatment attenuated serum IgG autoantibody levels in B6.Sle1.Sle3 mice (H-K). Each dot represents the autoantibody level of an individual mouse at the age of 2 mo (D0) or 4 mo (D60). AU = Arbitrary Unit. Serially diluted B6.Sle1.lpr sera were used for plotting a standard curve, and the highest standard was set as 100 AU. Student’s t-test was used to assess statistical significance.

CDDO-Me inhibited selected signaling axes in splenic B cells (A) or T cells (B) of B6.Sle1.Sle3 mice (female, n = 10 each). At the end of the 60-day CDDO-Me treatment period (i.e., all mice were at 4 mo age), splenic B or T cells were purified using magnetic beads, and cell lysates were examined for signaling status by western blot. Quantification of western blot results was done with ImageJ software (NIH) and plotted in the bar charts. Each bar is representative of band intensities observed in 10 mice. Student’s t-test was used to assess statistical significance. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

The Nrf2 signaling pathway was upregulated in the kidneys of B6.Sle1.Sle3 mice after CDDO-Me treatment. Kidneys were collected from both CDDO-Me-treated and placebo-treated B6.Sle1.Sle3 mice (female, n = 4-5 each), and kidney lysates were used for western blots. Quantification of western blot results was done with ImageJ (NIH) and plotted in the bar charts; each bar is representative of band intensities observed in 4-5 mice. Student’s t-test was used to assess statistical significance. (***, p < 0.001)

CDDO-Me ameliorated disease in another lupus mouse model-MRL.lpr. After 2 mo of treatment, splenomegaly was significantly reduced in CDDO-Me treated mice, compared to the water-treated group or sesame-treated group (N = 8 per group). Significantly reduced spleens were also noted in the dexamethasone-treated group (A). Compared to water-treated or sesame-treated mice, anti-dsDNA antibody levels (B), proteinuria and GN score (C) were also significantly decreased after CDDO-me treatment. Compared to the placebo group, the phosphorylation of MEK and Erk1,2 were reduced while Nrf2 was increased in both splenic B cells and T cells after CDDO-Me treatment.

CDDO-Me treatment significantly reduced cellular ROS in both splenic CD4+ T cells and CD11b+ cells but not B220+ B cells isolated from 4-month-old MRL.lpr mice (A-C). The reduction of ROS positively correlates with the decreased phosphorylation of Erk1,2 and MEK (D-E), but negatively correlates with the increase in Nrf2.